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Biomedical subjects

Y H Yang

Publications and source records attributed to Y H Yang.

At least 163 records · Page 9Linked to original sources
PubMed1983

Monoclonal antibodies for carcinoembryonic antigen and related antigens as a model system: a systematic approach for the determination of epitope specificities of monoclonal antibodies.

A systematic approach for the determination of epitope specificities of monoclonal antibodies to a complex antigen system is described. After initial screening to identify antigen-binding monoclonal antibodies, one or more of the clones are isolated by limiting dilution cloning, grown in ascites, and the resulting antibodies secreted into the ascitic fluid are affinity purified on Sepharose-bound protein A, radiolabeled, and cross-compared with antibodies from other clones by a solid-phase competitive immunoassay. In this work, BALB/c mice were immunized with either purified carcinoembryonic antigen (CEA) or the CEA-producing cell line HC 84S. Spleen cells were fused with the mouse myeloma cell line Sp2/0-Ag14. The supernatants from 25 hybrids showed a significant binding of 125I-CEA (greater than or equal to 15%). Nine hybrids were cloned, resulting in 33 different clones. The antibodies produced by the different cloned hybrids and the remaining uncloned hybrids recognized a total of five different epitopes on CEA. All of the epitopes reside on the protein moiety of the molecule as determined by antibody binding to deglycosylated CEA. The monoclonal antibodies with five different epitope specificities were reacted with tissue sections of normal and cancerous tissues and with peripheral blood smears. Each of the five monoclonal antibodies reacted with tissue sections from colonic, gastric, lung, and mammary carcinomas, as well as from a benign colonic polyp and a resection margin from a colonic carcinoma. Four monoclonals reacted with normal liver tissue. Granulocytes in peripheral blood smears bound three antibodies strongly and one antibody weakly, and one antibody was not bound. One monoclonal antibody that reacted with normal liver tissue was not bound by granulocytes. The ability of these five monoclonal antibodies to differentially detect three different CEA-related antigens in normal and malignant tissues may have clinical utility.

Animals↗
PubMed1982

Protein A binding assay for the identification of HLA antigens on peripheral blood lymphocytes by monoclonal antibodies: application to HLA B27.

Splenocytes from mice immunized with purified, papain-solubilized HLA B27 antigen and/or human lymphocytes bearing the B27 specificity were fused with myeloma cell lines NSI or Sp2. The screening strategy employed a protein A binding assay in which various target cells were used. First, the hybrid cell supernatants were screened against B lymphocyte cell lines of known HLA specificities and the Daudi cell line, which does not express HLA-A, B, or C antigens. Second, a panel of PBLs were used as target cells. It was necessary to refine the protein A binding assay by preabsorbing the radiolabeled protein A with PBLs and by precoating the test wells with ovalbumin. Clones selected by these criteria were further tested by indirect immunoprecipitation and by inhibition of binding or microcytotoxicty to target call lines with purified HLA antigens or beta 2m. Forty-four clones were selected which showed varying degrees of specificity for allo- and nonallo-specific determinants and one clone was selected which was specific for beta 2m. Clone 27M1 which was previously shown to be specific or HLA-B27 as judged by conventional microcytotoxicity testing (Grumet et al., Lancet No. 8239, II:174, 1981) was compared with other clones using the above parameters for evaluation. Antibody from clone 27M1 showed preferential binding to B27 positive cell lines and PBLs, lesser binding to B7 positive target cells, and no binding to B40 positive target cells. Purified B27 antigen (papain) from two sources including the B27 target cell line, was able to inhibit the binding of antibodies from 27M1 to target cells. The extension of the protein A binding assay to PBLs has made it possible to more accurately quantitate the binding or inhibition of binding of antibodies to panels of PBLs.

Animals↗
PubMed1979

Reversion of Kirsten sarcoma virus transformed human cells: elimination of the sarcoma virus nucleotide sequences.

The virus-specific nucleotide sequences in the RNA and DNA of a Kirsten mouse sarcoma virus (Ki-MSV)-transformed non-producer human osteosarcoma cell clone and two subclones of these cells that reverted to a normal phenotype have been analysed by hybridization of sarcoma virus-specific complementary DNA (cDNA) to cellular RNA or DNA. Whereas the transformed clone had acquired de novo Ki-MSV sequences in the RNA and DNA of the cells, both the revertant cell lines seemed to have lost most or all of this information from the cellular nucleic acids. The DNA from the revertant cells lacked the sequences represented either in the Ki-MSV-specific cDNA or in the total cDNA of the leukaemia-sarcoma virus complex. Thus, the reversion of the virus-transformed human cells to normal morphology is associated with the loss of most or all of the proviral sequences from the cellular DNA.

Base Sequence↗
PubMed1976

Adenosine triphosphate sulfurylase from penicillium chrysogenum. Steady state kinetics of the forward and reverse reactions.

The kinetic mechanism of ATP sulfurylase was established from initial velocity, product inhibition, and dead-end inhibition studies. In the forward direction, the reaction is steady state ordered, with MgATP=A, sulfate=B, MgPP1=P, and APS=Q.KmA=0.38 mM, Kia=0.71 mM, KmB=0.50 mM. Nitrate and chlorate are competive with sulfate and uncompetitive with MgATP. KiNO3-=0.25 mM; KiC1O3-= 0.15 mM. AMP and various MgATP analogs are competitive with MgATP and mixed-type inhibitors with respect to SO42-. The Ki for AMP is 0.55 mM. The reaction is rapid equilibrium ordered in the reverse direction with Kiq=0.3 to 1.0 muM and Kmp=0.65 muM. Adenosine 5'-phosphosulfate (APS) exhibits competitive substrate inhibition (KIQ=0.3 mM). The ratio Vmaxf/Vmaxr is 0.018. In the forward direction the ratio VmaxMoO42-/VmaxSO42- is 20. The Keq at pH 8.0 and 30 degrees calculated from the Haldane equation is 6 X 10(-9) to 3.3 X 10(-8) (depending on the Kiq value chosen). The experimental Keq is about 2.5 X 10(-9). The fact that Vmax/Vmaxr is about 1 million times greater than Keq is consistent with the assumed physiological role of the enzyme (APS synthesis). The mechanistic basis of the ordered binding sequence was probed by multiple inhibition analysis. Dead-end inhibitors competitive with MgATP (such as free ATP, Mg alpha,beta-methylene ATP, CrATP, and CaATP) do not induce substrate inhibition by sulfate or alter the inhibition patterns displayed by nitrate. This result suggests (but does not prove) that catalytic action on MgATP must precede the formation of the sulfate binding site.

Adenosine Triphosphate↗